Date of Award

Spring 6-2023

Document Type


Degree Name

Master of Science (MS)


Forensic Science



First Advisor or Mentor

Marta Concheiro-Guisan

Second Reader

Ana Pego

Third Advisor

Eduardo de Campos


The United States has been experiencing a nationwide public health crisis since introducing prescription opioids in the 1990s. A central problem with opioids is their highly addictive nature causing a dose-dependent respiratory depression leading to overdoses and deaths. Drug overdose deaths have risen in the past two decades, with opioids being the dominant driver of this epidemic. Given the circumstances, accurate and efficient confirmatory analysis of opioids in urine is essential to monitoring patients’ compliance and preventing drug diversion in clinical and forensic settings. The development of recombinant β-glucuronidases allows for the hydrolysis of glucuronidated opioids using a shorter incubation time and temperature, facilitating high-throughput forensic and clinical laboratories. Our goal was to optimize and evaluate the enzymatic hydrolysis procedure of seven opioid glucuronide metabolites in urine, including morphine-3-glucuronide, morphine-6-glucuronide, codeine-6-glucuronide, hydromorphone-glucuronide, oxymorphone-glucuronide, norbuprenorphine-glucuronide, and buprenorphine-glucuronide, using four different recombinant β-glucuronidase enzymes, BG-Turbo® and B-One® (Kura Biotech), and 04-RT-005 IMCSzyme® and 04-E1F-010 IMCSzyme®. We evaluated different volumes of enzyme (from 5 to 200 μL), temperatures (from room temperature to 60°C), and incubation times (10 to 60 mins). The most efficient enzymes included B-One® and 04-RT-005 IMCSzyme® recombinant β-glucuronidases providing a complete hydrolysis of opioids (>90%) that includes morphine-6-glucuronide and codeine-6-glucuronide using an incubation of 15 mins at room temperature. The optimized conditions for each β-glucuronidase enzymes were applied to 28 authentic cases.

Available for download on Thursday, May 22, 2025